PROJECT SUMMARY Streptococcus agalactiae, commonly referred to as Group B Streptococcus (GBS), is a leading cause of neonatal meningitis and sepsis worldwide as well as an agent of invasive disease in both pregnant and non-pregnant adults. GBS neonatal disease manifests as early onset, defined as disease within the first six days after birth, or late onset, defined as disease occurring after the first week of life; both of which are life threatening. In some instances, early onset disease is preventable with intrapartum antibiotic prophylaxis; however, this treatment strategy is not practical for low- and middle-income countries nor does it prevent late onset disease or the ~3.5 million preterm and 60,000 stillbirths attributed to GBS each year. Molecularly, GBS produces one of ten different capsular polysaccharides, five of which (serotype Ia, Ib, II, III, and V) are associated with 97% of all invasive neonatal GBS disease events. In addition, previous studies have demonstrated that placental transfer of maternal antibodies are able protect neonates from invasive GBS infection. Therefore, a vaccine targeting these five serotypes is of high societal and commercial value. In order to make an efficacious vaccine targeting GBS capsules, the polysaccharide must be covalently linked to an immunogenic carrier protein generating what?s termed a conjugate vaccine. Conjugate vaccines are considered some of the most effective vaccines to date, as they are highly protective and generate immunological memory across all age groups. However, their synthesis is complex, costly, and not conducive for all polysaccharides, which has hindered development of novel conjugate vaccines against life threatening bacteria like GBS. As an alternative manufacturing platform, VaxNewMo is developing conjugate vaccines using an innovative in vivo conjugation technology, which eliminates the dependency on intricate chemical conjugation methods currently employed to synthesize these vaccines. Using VaxNewMo?s proprietary bioconjugating enzyme technology, we will therefore generate the most broadly covering GBS vaccine against five of the most prevalent GBS serotypes causing 97% of all neonatal invasive disease events. The proposed research in this phase I application will focus on (Aim 1) developing five glycoengineered strains of E. coli for the scalable, recombinant expression of GBS capsular polysaccharides in conjunction with VaxNewMo?s conjugating enzyme technology, generating the first bioconjugate vaccine against GBS. Subsequently (Aim 2) we will demonstrate that a monovalent GBS bioconjugate vaccine is immunogenically non-inferior to a traditionally prepared chemical GBS conjugate vaccine. We will also demonstrate the versatility of VaxNewMo?s bioconjugating platform by providing preliminary immunogenicity data on a pentavalent GBS bioconjugate. Last, we will test for correlates of immunity by determining bacterial burden and/or survival of newborn pups infected with GBS born from vaccinated mice. Our next step for Phase II funding will be to develop and optimize a purification protocol for large scale quantities of GBS bioconjugate vaccines and provide sufficient safety, tolerability and immunogenicity data for Fast Track FDA approval.